Methods for treating bone gap defects

ABSTRACT

The invention provides a method of enhancing bone gap defect healing involving administering a sclerostin inhibitor.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to methods of using sclerostininhibitors to enhance bone gap defect healing.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: ASCII (text) file named“46346_PCTSeqListing.txt,” 800,882 bytes, created on Jul. 30, 2012.

INCORPORATION BY REFERENCE

The following applications are hereby incorporated by reference in theirentirety: U.S. patent application Ser. No. 11/410,540, filed Apr. 25,2006, which claims priority to U.S. Provisional Patent Application No.60/792,645, filed Apr. 17, 2006, U.S. Provisional Patent Application No.60/782,244, filed Mar. 13, 2006, U.S. Provisional Patent Application No.60/776,847, filed Feb. 24, 2006, and U.S. Provisional Patent ApplicationNo. 60/677,583, filed May 3, 2005; and U.S. patent application Ser. No.11/411,003 (issued as U.S. Pat. No. 7,592,429), filed Apr. 25, 2006,which claims priority to U.S. Provisional Patent Application No.60/792,645, filed Apr. 17, 2006, U.S. Provisional Patent Application No.60/782,244, filed Mar. 13, 2006, U.S. Provisional Patent Application No.60/776,847, filed Feb. 24, 2006, and U.S. Provisional Patent ApplicationNo. 60/677,583, filed May 3, 2005. The following applications also arehereby incorporated by reference: U.S. patent application Ser. No.12/212,327, filed Sep. 17, 2008, which claims priority to U.S.Provisional Patent Application No. 60/973,024, filed Sep. 17, 2007; andU.S. patent application Ser. No. 12/811,171, filed Jun. 29, 2010, whichis a U.S. National Phase Application pursuant to 35 U.S.C. § 371 ofInternational Patent Application No. PCT/US08/86864, filed on Dec. 15,2008, which claims priority to U.S. Provisional Patent Application No.61/013,917, filed Dec. 14, 2007.

BACKGROUND OF THE INVENTION

Mammalian bone tissue has a remarkable ability to regenerate and therebyrepair injuries and other defects. For example, bone growth is generallysufficient to bring about full recovery from most simple and hairlinefractures. Unfortunately, however, there are many injuries, defects orconditions where bone growth is inadequate to achieve an acceptableoutcome. For example bone regeneration generally does not occurthroughout large voids or spaces. Therefore, fractures cannot healunless the pieces are in close proximity. If a significant amount ofbone tissue was lost as a result of the injury, the healing process maybe incomplete, resulting in undesirable cosmetic and/or mechanicaloutcomes. This is often the case with non-union fractures or with boneinjuries resulting from massive trauma. Tissue growth is also generallyinadequate in voids and segmental gaps in bone caused, for example, bysurgical removal of tumors or cysts. In other instances, it may bedesirable to stimulate bone growth where bone is not normally found,i.e., ectopically. Spine fusion to relieve lower back pain where two ormore vertebrae are induced to fuse is one example of desirable ectopicbone formation.

SUMMARY OF THE INVENTION

The invention is directed to methods of using a sclerostin inhibitor totreat humans with bone gap defects. In one aspect, described herein is amethod of treating a bone gap defect in a subject, wherein the methodcomprises administering to the subject an effective amount of asclerostin inhibitor (e.g., an anti-sclerostin antibody), optionally ata weekly dose from about 1 mg/kg to about 50 mg/kg per week, wherein thesclerostin inhibitor is administered over a treatment period lasting atleast about 11 weeks. In one embodiment, the sclerostin inhibitor isadministered once a week for the duration of the treatment period. Inanother embodiment, the sclerostin inhibitor is administered once everytwo weeks for the duration of the treatment period. Alternatively, thesclerostin inhibitor is administered twice per week.

The treatment period can be at least about 11 weeks, 12 weeks, 3 months,13 weeks, 14 weeks, 15 weeks, 16 weeks, 4 months, 17 weeks, 18 weeks, 19weeks, 20 weeks, 5 months, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 6months, 25 weeks, 26 weeks, 27 weeks 28 weeks, 7 months, 29 weeks, 30weeks, 31 weeks or longer (e.g., 8 months, 9 months, 10 months, 11months, 1 year, 15 months, 18 months or longer). In some embodiments,the treatment period is about 20-32 weeks, or about 5-8 months. In someembodiments, the treatment period is no more than about 28 weeks. Insome embodiments, the treatment period is about 1 year. In some or anyembodiments, the treatment period is no more than about 18 months.

The bone gap defect for treatment by the methods described hereinincludes any fracture comprising a gap between two segments of bone(e.g., a gap of at least about 1 mm between two segments of bone). Insome or any embodiments, the gap is at least about 2 mm, at least about3 mm, at least about 4 mm, at least about 5 mm, at least about 6 mm, atleast about 7 mm, at least about 8 mm, at least about 9 mm, or at leastabout 1 cm or more. In some or any embodiments, the gap is about 5 mm to1 cm, or up to 1 cm.

Exemplary bone gap defects include, but are not limited to, a comminutedfracture, a non-union fracture, a segmental skeletal defect, surgicallycreated bone defects, surgically treated bone defects, and bone defectscreated from traumatic injury to the bone or disease (including, but notlimited to, arthritis, tumor removal (resection) or infection removal).In some or any embodiments, the bone gap defect is produced by removalof infected sections of bone or the removal of cancer from the bone dueto bone cancers including, but not limited to, osteosarcoma, Ewing'ssarcoma, chondrosarcoma, malignant fibrous histiocytoma, fibrosarcoma,and chordoma. In some or any embodiments, the bone gap defect is adevelopmental deformity, e.g., due to a genetic defect.

In some or any embodiments, the bone gap defect is produced by removalof sections of bone containing a benign tumor. Exemplary benign bonetumors include, but are not limited to, osteoma, osteoid osteoma,osteoblastoma, osteochondroma, enchondroma, chonrdomyxoid fibroma,aneurysmal bone cyst, unicameral bone cyst, fibrous dysplasia of boneand giant cell tumor of the bone.

The subject to which the sclerostin inhibitor (e.g., anti-sclerostinantibody) is administered is optionally suffering from a bone-relateddisorder selected from the group consisting of achondroplasia,cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher'sDisease, hypophosphatemic rickets, Marfan's syndrome, multiplehereditary exotoses, neurofibromatosis, osteogenesis imperfecta,osteopetrosis, osteopoikilosis, sclerotic lesions, pseudoarthrosis,pyogenic osteomyelitis, periodontal disease, anti-epileptic drug inducedbone loss, primary and secondary hyperparathyroidism, familialhyperparathyroidism syndromes, weightlessness induced bone loss,osteoporosis in men, postmenopausal bone loss, osteoarthritis, renalosteodystrophy, infiltrative disorders of bone, oral bone loss,osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis,metabolic bone diseases, mastocytosis, sickle cell anemia/disease, organtransplant related bone loss, kidney transplant related bone loss,systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenilearthritides, thalassemia, mucopolysaccharidoses, Fabry Disease, TurnerSyndrome, Down Syndrome, Klinefelter Syndrome, leprosy, Perthe'sDisease, adolescent idiopathic scoliosis, infantile onset multi-systeminflammatory disease, Winchester Syndrome, Menkes Disease, Wilson'sDisease, ischemic bone disease (such as Legg-Calve-Perthes disease andregional migratory osteoporosis), anemic states, conditions caused bysteroids, glucocorticoid-induced bone loss, heparin-induced bone loss,bone marrow disorders, scurvy, malnutrition, calcium deficiency,osteoporosis, osteopenia, alcoholism, chronic liver disease,postmenopausal state, chronic inflammatory conditions, rheumatoidarthritis, inflammatory bowel disease, ulcerative colitis, inflammatorycolitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy,diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroiddisorders, Cushing's disease, acromegaly, hypogonadism, immobilizationor disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis,osteomalacia, bone loss associated with joint replacement, HIVassociated bone loss, bone loss associated with loss of growth hormone,bone loss associated with cystic fibrosis, chemotherapy-associated boneloss, tumor-induced bone loss, cancer-related bone loss, hormoneablative bone loss, multiple myeloma, drug-induced bone loss, anorexianervosa, disease-associated facial bone loss, disease-associated cranialbone loss, disease-associated bone loss of the jaw, disease-associatedbone loss of the skull, bone loss associated with aging, facial boneloss associated with aging, cranial bone loss associated with aging, jawbone loss associated with aging, skull bone loss associated with aging,and bone loss associated with space travel. In one embodiment, thesubject is undergoing oral or maxillofacial surgery.

In some or any embodiments, the sclerostin inhibitor (e.g.anti-sclerostin antibody) is administered in combination with the use ofmaterials that promote the regrowth of bone such as bone graft, bonedust, bone chips, demineralized bone matrix, bone scaffolds, prosthesis,metal stabilizers, or bone scaffold substances comprising one or more ofpolymers, ceramics, cement and calcium phosphates-based bone-graftsubstitutes. Many variations of such materials are known in the art.

In some or any embodiments, the sclerostin inhibitor (e.g.,anti-sclerostin antibody) is administered along with a secondbone-enhancing therapeutic for the treatment of decreased bone mineraldensity or bone fracture. Many therapeutics of this type are known inthe art. In some embodiments, the bone-enhancing therapeutic is selectedfrom the group consisting of an anti-resorptive drug, a bone-formingagent, an estrogen receptor antagonist (including, but not limited to,raloxifene, bazedoxifene and lasofoxifene) and a drug that has aninhibitory effect on osteoclasts. In some embodiments, theanti-resorptive drug includes, but is not limited to, parathyroidhormone, a bisphosphonate (including, but not limited to, alendronate,risedronate, ibandronate and zoledronate), an estrogen or estrogenanalogue, a selective estrogen receptor modulator (SERM) and a calciumsource, Tibolone, calcitonin, a calcitriol and hormone replacementtherapy. In some embodiments, the bone-enhancing agent includes, but isnot limited to parathyroid hormone (PTH) or a peptide fragment thereof,PTH-related protein (PTHrp), bone morphogenetic protein, osteogenin,NaF, a PGE₂ agonist, a statin, an anti-DKK1 antibody or inhibitor, ananti-RANK ligand (RANKL) antibody or RANKL inhibitor, strontiumranelate, vitamin D, or a vitamin D derivative or mimic thereof. In someembodiments, the bone-enhancing agent is Forteo® (Teriparatide, orrecombinant human parathyroid hormone 1-34) or Preotact® (parathyroidhormone). In some or any embodiments, the bone-enhancing agent isProtelos®.

In any of the embodiments disclosed herein, the sclerostin inhibitor isoptionally a sclerostin binding agent (e.g., an anti-sclerostinantibody). The use of sclerostin binding agents disclosed in U.S. PatentPublication No. 20070110747, e.g., in any of the methods disclosedherein or for preparation of medicaments for administration according toany of the methods disclosed herein, is specifically contemplated. Oneor more doses of the sclerostin inhibitor are administered in an amountand for a time effective to enhance gap defect healing at the fracturesite. One or more doses of sclerostin inhibitor can comprise betweenabout 1 to about 50 milligrams (e.g., between about 10 and about 50milligrams), or about 1 to about 100 milligrams, of sclerostin inhibitorper kilogram of body weight (mg/kg). For example, the dose of sclerostininhibitor (e.g., anti-sclerostin antibody) may range from at least about1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg,about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10mg/kg, about 20 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg,about 28 mg/kg, about 29 mg/kg, about 30 mg/kg, about 31 mg/kg, about 32mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg, about 36 mg/kg,about 37 mg/kg, about 38 mg/kg, about 39 mg/kg, about 40 mg/kg, about 41mg/kg, about 42 mg/kg, about 43 mg/kg, about 44 mg/kg, about 45 mg/kg,about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, or about 49 mg/kg, orabout 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg,about 95 mg/kg, or up to about 100 mg/kg. Ranges between any and all ofthese endpoints are also contemplated, e.g. about 1 to about 3 mg/kg,about 1 to about 5 mg/kg, about 1 to about 10 mg/kg, about 1 to about 20mg/kg, about 1 to about 40 mg/kg, about 5 to about 30 mg/kg, or about 5to about 20 mg/kg. In some embodiments, the sclerostin inhibitor isadministered shortly after the fracture (e.g., within 30 minutes, within1 hour, within 2 hours, within 6 hours, within 12 hours or within 24hours of the fracture). In other embodiments, the inhibitor isadministered within 1 day of the fracture, within 3 days of thefracture, within 5 days of the fracture, within 7 days of the fracture,within two weeks of the fracture, wherein the sclerostin binding agentis administered for a period of time that is at least 11 weekspost-fracture (e.g., 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks,16 weeks, 17 weeks, 18 weeks 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks 28 weeks, 29 weeks, 30weeks, 31 weeks or longer (e.g., 8 months, 9 months, 10 months, 11months, 1 year, 18 months or longer)).

Also described herein is the use of an effective amount of ananti-sclerostin antibody for treating a bone gap defect in a subject,for example, in any of the amounts described above, such as from about 1mg/kg to about 100 mg/kg, wherein one or more administrations of thesclerostin binding agent is carried out over a treatment period lastingat least 11 weeks (e.g., any of the time periods described above, suchas 12 weeks, 3 months, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 4 months,17 weeks, 18 weeks 19 weeks, 20 weeks, 5 months, 21 weeks, 22 weeks, 23weeks, 24 weeks, 6 months, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 7months, 29 weeks, 30 weeks, 31 weeks or longer (e.g., 8 months, 9months, 10 months, 11 months, 1 year, 15 months, 18 months or longer)).

The sclerostin inhibitor (e.g., anti-sclerostin antibody) may be alsoused in the preparation of a medicament for administration to a subjectwith a bone gap defect using any of the dosing and/or timing regimensdescribed herein. Thus, the invention also contemplates sclerostininhibitor for use according to any of the dosing and/or timing regimensdescribed herein. Optionally, the sclerostin inhibitor is presented in acontainer, such as a single dose or multidose vial. The inventionincludes a container comprising anti-sclerostin antibody or fragmentthereof and instructions for administering the antibody or fragmentthereof for treating a bone gap defect according to any of the dosingand/or timing regimens described herein.

In some embodiments, the anti-sclerostin antibody for use in the methodsdescribed herein binds to sclerostin of SEQ ID NO: 1, with an affinity(Kd) of less than or equal to 1×10⁻⁷M (or less than or equal to 1×10⁻⁸M,or less than or equal to 1×10⁻⁹M, or less than or equal to 1×10⁻¹⁰ M, orless than or equal to 1×10⁻¹¹M, or less than or equal to 1×10⁻¹² M).

In various embodiments, the anti-sclerostin antibody binds to asclerostin polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 1 and binds the sequence of SEQ ID NO: 6(CGPARLLPNAIGRGKWWRPSGPDFRC; corresponding to amino acids 86-111 of SEQID NO: 1). Alternatively or in addition, the anti-sclerostin antibodybinds to a sclerostin polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 1 and binds the sequence of at least one of SEQ IDNO: 2 (DVSEYSCRELHFTR; corresponding to amino acids 51-64 of SEQ ID NO:1), SEQ ID NO: 3 (SAKPVTELVCSGQCGPAR; corresponding to amino acids 73-90of SEQ ID NO: 1), SEQ ID NO: 4 (WWRPSGPDFRCIPDRYR; corresponding toamino acids 101-117 of SEQ ID NO: 1), SEQ ID NO: 5 (LVASCKCKRLTR;corresponding to amino acids 138-149 of SEQ ID NO: 1), SEQ ID NO: 70(SAKPVTELVCSGQC; corresponding to amino acids 73-86 of SEQ ID NO: 1),SEQ ID NO: 71 (LVASCKC; corresponding to amino acids 138-144 of SEQ IDNO: 1), SEQ ID NO: 72 (CRELHFTR; corresponding to amino acids 57-64 ofSEQ ID NO: 1), or SEQ ID NO: 73 (CIPDRYR; corresponding to amino acids111-117 of SEQ ID NO: 1) within SEQ ID NO: 1. For example, in oneaspect, the anti-sclerostin antibody binds a subregion of sclerostin ofSEQ ID NO: 1 comprising SEQ ID NOs: 2-5 (and/or SEQ ID NOs: 70-73),optionally in its native three-dimensional conformation. Optionally, theanti-sclerostin antibody binds a peptide consisting of one or more ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73 (e.g., apeptide consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQID NO: 5 or a peptide consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ IDNO: 72, and SEQ ID NO: 73).

In various aspects, the anti-sclerostin antibody is capable ofneutralizing human sclerostin in a MC3T3 cell-based mineralization assaywhen there is less than a 6-fold excess of moles of sclerostin bindingsites per well as compared to the number of moles of sclerostin perwell.

The anti-sclerostin antibody optionally has an IC₅₀ of 100 nM or less,or 75 nM or less, or 50 nM or less, or 25 nM or less for neutralizinghuman sclerostin in a cell-based assay, such as a bone specific alkalinephosphatase assay. Alternatively or in addition, the anti-sclerostinantibody has an IC₅₀ of 100 nM or less (e.g., 75 nM or less, or 50 nM orless) for neutralizing human sclerostin in a cell-based Wnt signalingassay in HEK293 cell lines, such as the Wnt assay involvingWnt1-mediated induction of STF reporter gene. Alternatively or inaddition, the anti-sclerostin antibody has an IC₅₀ of 500 nM or less(e.g., 250 nM or less, 150 nM or less, 100 nM or less, or 50 nM or less)for neutralizing human sclerostin in a BMP2-induced mineralization assayin MC3T3 cells.

In one embodiment, the anti-sclerostin antibody cross-blocks the bindingof at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3,Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14,Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24to sclerostin and/or is cross-blocked from binding to sclerostin by atleast one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4,Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15,Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24.

In some embodiments, the anti-sclerostin antibody comprises a CDR-H1 ofSEQ ID NO:245, a CDR-H2 of SEQ ID NO:246, a CDR-H3 of SEQ ID NO:247, aCDR-L1 of SEQ ID NO:78, a CDR-L2 of SEQ ID NO:79 and a CDR-L3 of SEQ IDNO:80.

In one embodiment, the anti-sclerostin antibody comprises heavy chainscomprising SEQ ID NO: 378 and light chains comprising SEQ ID NO: 376. Inanother embodiment, anti-sclerostin antibody has heavy chains of SEQ IDNO: 145 or SEQ ID NO: 392 and light chains of SEQ ID NO: 141.

In another embodiment, the anti-sclerostin antibody comprises CDRs ofSEQ ID NOs: 20-25 of International Patent Publication No. WO 2008/115732(SEQ ID NOs: 416-421), CDRs of SEQ ID NOs: 26-31 of International PatentPublication No. WO 2008/115732 (SEQ ID NOs: 422-427), CDRs of SEQ IDNOs: 32-37 of International Patent Publication No. WO 2008/115732 (SEQID NOs: 428-433), or CDRs of SEQ ID NOs: 4, 15, 26, 37, 48, and 59 ofInternational Patent Publication No. WO 2009/047356 (SEQ ID NOs: 443,454, 465, 476, 487, and 498, respectively). In yet another embodiment,the anti-sclerostin antibody comprises an amino acid sequence of atleast one of SEQ ID NOs: 135-143, 153-161, or 171-179 of InternationalPatent Publication No. WO 2010/130830 (SEQ ID NOs: 745-753, 763-771,781-789, respectively).

The invention is also described in the following exemplary embodiments:

1. A method for treating a bone gap defect in a subject comprisingadministering to the subject an effective amount of an anti-sclerostinantibody, optionally at a weekly dose from about 1 mg/kg to about 50mg/kg per week, wherein the sclerostin binding agent is administeredover a treatment period lasting at least 20 weeks.

2. The method of paragraph 1, wherein the treatment period lasts about28 weeks.

3. The method of paragraph 1, wherein the bone gap defect is selectedfrom the group consisting of a comminuted fracture, a non-union fracturea segmental skeletal defect, surgically created bone defects, surgicallytreated bone defects, and bone defects created from traumatic injury tothe bone or disease (including arthritis, developmental deformity, tumorremoval (resection) or infection removal).

4. The method of paragraph 3, wherein the bone gap defect is produced byremoval of infected sections of bone or the removal of cancer from thebone.

5. The method of paragraph 4, wherein the cancer is selected from thegroup consisting of neck cancer, head cancer, bone cancer and jawcancer.

6. The method of paragraph 5, wherein the bone cancer is selected fromthe group consisting of osteo sarcoma, Ewing's sarcoma, chondrosarcoma,malignant fibrous histiocytoma, fibrosarcoma, and chordoma.

7. The method of paragraph 3, wherein the bone gap defect is produced byremoval of a benign tumor from the bone.

8. The method of paragraph 7, wherein the benign bone tumor is selectedfrom the group consisting of osteoma, osteoid osteoma, osteoblastoma,osteochondroma, enchondroma, chonrdomyxoid fibroma, aneurysmal bonecyst, unicameral bone cyst, fibrous dysplasia of bone and giant celltumor of the bone.

9. The method of paragraph 4, wherein the cancer is multiple myeloma.

10. The method of paragraph 1, wherein the subject is also receiving abone graft, bone dust, bone chips, cartilage transplant, bone scaffolds,prosthesis, metal stabilizers, or bone scaffold substances comprisingone or more of polymers, ceramics, cement and calcium phosphates.

11. The method of paragraph 1, wherein the subject is undergoing oral ormaxillofacial surgery.

12. The method of paragraph 1, further comprising administering a secondbone-enhancing therapeutic selected from the group consisting ofparathyroid hormone, a bisphosphonate, a RANKL antibody and a DKK-1antibody.

13. The method of any one of paragraphs 1-12, wherein theanti-sclerostin antibody is administered in an amount of 30 mg/kg perweek.

14. The method of any one of paragraphs 1-13, wherein theanti-sclerostin antibody is administered once a week for the duration ofthe treatment period.

15. The method of any one of paragraphs 1-8, wherein treatment with theanti-sclerostin antibody does not result in a substantial increase incortical porosity in bone of the subject.

16. The method of any one of paragraphs 1-5, wherein the anti-sclerostinantibody is administered subcutaneously.

17. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody is an immunoglobulin comprising a heavy chainand a light chain.

18. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody is an antibody or fragment thereof thatdemonstrates a binding affinity for sclerostin of SEQ ID NO: 1 of lessthan or equal to 1×10⁻⁷ M.

19. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody neutralizes human sclerostin in a MC3T3cell-based mineralization assay when there is less than a 6-fold excessof moles of sclerostin binding sites per well as compared to the numberof moles of sclerostin per well.

20. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody has an IC₅₀ of 100 nM or less, 50 nM or less,or 25 nM or less for neutralizing human sclerostin in a cell-basedassay, such as a bone specific alkaline phosphatase assay.

21. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody has an IC₅₀ of 100 nM or less for neutralizinghuman sclerostin in a cell-based Wnt signaling assay in HEK293 celllines.

22. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody has an IC₅₀ of 500 nM or less for neutralizinghuman sclerostin in a BMP2-induced mineralization assay in MC3T3 cells.

23. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody binds to a sclerostin polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 1, wherein saidanti-sclerostin antibody binds to the sequence of SEQ ID NO: 6.

24. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody binds to a sclerostin polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 1, wherein saidanti-sclerostin antibody binds to the sequence of at least one of SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

25. The method of any one of paragraphs 1-15, wherein theanti-sclerostin antibody binds to a sclerostin polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 1, wherein saidanti-sclerostin antibody binds to the sequence of at least one of SEQ IDNO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

26. The method of any of the paragraphs 1-25, where the anti-sclerostinantibody cross-blocks the binding of at least one of antibodies Ab-A,Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9,Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19,Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin and/or iscross-blocked from binding to sclerostin by at least one of antibodiesAb-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8,Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18,Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, and Ab-24.

27. The method of paragraph 26, wherein the anti-sclerostin antibodycomprises a CDR-H1 of SEQ ID NO:245, a CDR-H2 of SEQ ID NO:246, a CDR-H3of SEQ ID NO:247, a CDR-L1 of SEQ ID NO:78, a CDR-L2 of SEQ ID NO:79 anda CDR-L3 of SEQ ID NO:80.

28. The method of paragraph 27, wherein anti-sclerostin antibodycomprises heavy chains comprising SEQ ID NO: 378 and light chainscomprising SEQ ID NO 376.

29. The method of paragraph 27, wherein anti-sclerostin antibody hasheavy chains of SEQ ID NO: 145 or SEQ ID NO: 392 and light chains of SEQID NO: 141.

30. The method of any of the paragraphs 1-25, wherein theanti-sclerostin antibody comprises CDRs of SEQ ID NOs: 20-25 ofInternational Patent Publication No. WO 2008/115732 (SEQ ID NOs:416-421), CDRs of SEQ ID NOs: 26-31 of International Patent PublicationNo. WO 2008/115732 (SEQ ID NOs: 422-427), or CDRs of SEQ ID NOs: 32-37of International Patent Publication No. WO 2008/115732 (SEQ ID NOs:428-433).

31. The method of any of the paragraphs 1-25, wherein theanti-sclerostin antibody comprises CDRs of SEQ ID NOs: 4, 15, 26, 37,48, and 59 of International Patent Publication No. WO 2009/047356 (SEQID NOs: 443, 454, 465, 476, 487, and 498, respectively).

32. The method of any of the paragraphs 1-25, wherein theanti-sclerostin antibody comprises the amino acid sequence of at leastone of SEQ ID NOs: 135-143, 153-161, or 171-179 of International PatentPublication No. WO 2010/130830 (SEQ ID NOs: 745-753, 763-771, 781-789,respectively).

The foregoing summary is not intended to define every aspect of theinvention, and additional aspects are described in other sections, suchas the Detailed Description. The entire document is intended to berelated as a unified disclosure, and it should be understood that allcombinations of features described herein are contemplated, even if thecombination of features are not found together in the same sentence, orparagraph, or section of this document. With respect to aspects of theinvention described or claimed with “a” or “an,” it should be understoodthat these terms mean “one or more” unless context unambiguouslyrequires a more restricted meaning. The term “or” should be understoodto encompass items in the alternative or together, unless contextunambiguously requires otherwise. If aspects of the invention aredescribed as “comprising” a feature, embodiments also are contemplated“consisting of” or “consisting essentially of” the feature.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart listing amino acid sequences and sequence identifiersfor amino acid sequences of various anti-sclerostin antibodies describedherein. The sequence identifiers refer to amino acid sequences providedin the Sequence Listing submitted herewith. The amino acid sequencesalso are set forth in U.S. Patent Publication No. 2007/0110747 orInternational Patent Publication Nos. WO 2008/115732, WO2009/047356, orWO 2010/130830, hereby incorporated by reference.

FIGS. 2A and 2B provide graphs which illustrate that administration ofthe anti-sclerostin antibody resulted in increased cortical area andcortical thickness in the femoral diaphysis of healthy cynomologusmonkeys after 28 weeks of treatment compared to animals that did notreceive the antibody treatment.

FIGS. 3A and 3B provide graphs which illustrate that administration ofthe anti-sclerostin antibody resulted in increased periosteal andendocortical bone formation rate in the femoral diaphysis of healthycynomologus monkeys after 28 weeks of treatment compared to animals thatdid not receive the antibody treatment.

FIG. 4 provides a graph which illustrates that administration of theanti-sclerostin antibody did not substantially increase corticalporosity in the femoral diaphysis of healthy cynomologus monkeys after28 weeks of treatment compared to animals that did not receive theantibody treatment.

DETAILED DESCRIPTION OF THE INVENTION

The invention is predicated, at least in part, on the discovery thatsclerostin inhibitors enhance healing of bone gap defects. In thisregard, the invention provides a method of treating a segmental skeletaldefect or a non-union fracture. The method comprises administering to asubject (e.g., a mammal, such as a human) one or more doses of asclerostin inhibitor, such as sclerostin binding agent (e.g., ananti-sclerostin antibody), during a treatment period of, e.g., at least11 weeks. The materials and methods of the invention are superior toexisting therapies whose therapeutic efficacy is limited and requireextended recovery time.

The terms “bone gap defect” and “segmental skeletal defect” are usedsynonymously herein and refer to a gap between two segments of bone(e.g., a gap of at least 1 mm).

Administration of the sclerostin inhibitor enhances or accelerates bonegap defect healing, thereby “treating” the bone gap defect. “Enhancing”bone healing means mediating a level of bone healing beyond (i.e.,greater than) the level of bone healing experienced in subjects (e.g.,mammals, such as humans) not administered the sclerostin inhibitor(i.e., control subjects). Bone healing is evidenced by, for example,bridging status, improved bone volume, improved bone mineral content anddensity within the fracture gap (i.e., formation of bridging bone),mature bone callus, improved bone strength (optionally accompanied by amedically-acceptable level of bone stiffness), or improved patient useof the affected area. By “improved” is meant an increase or decrease (asdesired) in the measured parameter. The increase can be a return, inwhole or in part, of the measured parameter to baseline level (e.g., thelevel prior to the bone gap defect), to values provided in normativedatabases used in the art, or to the contralateral functional level(e.g., return, in whole or in part, to the functional capabilities of,for example, the contralateral limb). In some cases, the increase can bean improvement beyond baseline level. If desired, the measuredparameters in patients administered one or more doses of the sclerostininhibitor can be compared to the same parameters in fracture patients(optionally age and gender matched) not administered the sclerostininhibitor to further analyze the efficacy of the methods describedherein.

Formation of bridging bone, bone mineral content and bone density,and/or mature honey callus at the site of bone defect may be measuredusing radiography (e.g., radiographic absorptometry), single- and/ordual-energy X-ray absorptometry, quantitative computed tomography (QCT),ultrasonography, radiography (e.g., radiographic absorptometry), andmagnetic resonance imaging. In some embodiments, the sclerostininhibitor (e.g., sclerostin binding agent) may be administered at a doseand for a time period effective to increase bridging bone formation,formation of bony callus, or bone density (or volume) at the defect siteby at least about 5% (about 6%, about 7%, about 8%, or about 9%). Insome embodiments, bridging bone formation, formation of bony callus, orbone density at the defect site is increased by at least about 10%(e.g., at least about 10%, at least about 12%, at least about 15%, atleast about 18%, at least about 20%, or at least about 22%). In otherembodiments, bridging bone formation, formation of bony callus, or bonedensity at the defect site is increased by the sclerostin inhibitor atleast about 25% (e.g., at least about 26% or at least about 28%). In yetother embodiments, bridging bone formation, formation of bony callus, orbone density at the defect site is increased at least about 30% (e.g.,at least about 32%, at least about 35%, at least about 38%, or at leastabout 40%) or at least about 50% (e.g., at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or about 100%). Theincrease or re-establishment of bridging bone formation can bedetermined at 1 week, 2 weeks, 3 weeks, or 4 weeks following the initialadministration of sclerostin inhibitor. Alternatively, the bone densitylevel can be determined after the treatment period ends (e.g., 1 week, 2weeks, 3 weeks, or 4 weeks after the treatment period ends). In oneaspect, the method reduces the amount of time required to establish adesired level of bone formation, bone volume, bony callus, or bonedensity (e.g., any percent increase in bone formation, bone mineraldensity, bony callus, or bone volume described herein) compared to ageand gender-matched patients that do not receive the sclerostininhibitor, thereby reducing recovery time for a subject. For example, inone embodiment, the sclerostin inhibitor reduces the amount of timerequired to increase bone density or volume at the defect site at leastabout 10% (e.g., at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, or atleast about 50%).

Functional, quality of life parameters indicative of bone healinginclude, but are not limited to, recovery of strength and load-bearingcapacity, decreased pain and use of pain medication, and improvedoccupational status. Administration of one or more doses of a sclerostininhibitor, as described herein, accelerates improvement of functional,quality of life parameters associated with fractures in a statisticallysignificant manner in the patient population tested. In certain aspects,the method reduces recovery time in the patient administered one or moredoses of sclerostin inhibitor by at least 10% (e.g., at least 20%, atleast 30%, at least 40%, at least 50%, or at least 65%) compared torecovery time in patients that do not receive the sclerostin inhibitor.“Recovery” can be estimated using any of a number of rehabilitationoutcome measurements, such as the FIM instrument motor score for hipfractures (Munin et al., Arch. Phys. Med. Rehabil., 86:367-372 (2005)),the Olerud-Molander Ankle Score (OMAS) and SF-12 questionnaire for anklefracture (Shah et al., Injury, 38(11):1308-1312 (2003)), and KneeSociety Scoring for knee replacements (Insall et al., ClinicalOrthopaedics, 248:13-14 (1989)).

In some embodiments, one or more doses of a sclerostin inhibitor, suchas a sclerostin binding agent (e.g., an anti-sclerostin antibody) isadministered to a human over the course of a treatment period comprising11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 31 weeks, 8 months, 9 months, 10 months, 11 months, 1 year, 18months or longer. A “treatment period” begins upon administration of afirst dose of sclerostin inhibitor (e.g., anti-sclerostin antibody) andends upon administration of a final dose of sclerostin inhibitor. A doseof sclerostin inhibitor may be administered multiple times per week, ifdesired. In one embodiment, the treatment period comprises at least 11weeks. In some embodiments, the treatment period lasts 28 weeks. Inother embodiments, the treatment period lasts 1 year. Alternatively orin addition, the treatment period lasts no more than 18 months. Indeed,one or more administrations of a pharmaceutical composition comprisingthe sclerostin inhibitor may be carried out over a treatment ortherapeutic period lasting no more than 18 months, less than 1 year, nomore than 8 months, no more than 28 weeks, or no more than 20 weeks. Inone embodiment, the treatment period is about 28 weeks and, yieldssignificant improvement in healing parameters, such as (but not limitedto) bone formation, bone strength (e.g., maximum load-bearing capacitybefore experiencing pain), bone volume, no substantial increase incortical porosity, bridging limb function, and/or recovery time, whencompared to untreated fractures. In addition, in one aspect, thetreatment period begins soon after a bone gap defect is detected, e.g.,within 30 minutes, within 1 hour, within 2 hours, within 6 hours, within12 hours or within 24 hours of the defect. In other embodiments, theinhibitor is administered within 1 day of the bone defect, within 3 daysof the bone defect, within 5 days of the bone defect, within 7 days ofthe bone defect, or within two weeks of the bone defect, wherein thesclerostin binding agent is administered for a period of time that is atleast 11 weeks post-bone defect (e.g., 11 weeks, 12 weeks, 13 weeks, 14weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks 19 weeks, 20 weeks, 21weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks 28weeks, 29 weeks, 30 weeks, 31 weeks or longer (e.g., 8 months, 9 months,10 months, 11 months, 1 year, 18 months or longer)).

The sclerostin binding agent (e.g., anti-sclerostin antibody) isadministered in an amount that promotes, enhances, or accelerateshealing of the bone gap defect. The dose of sclerostin binding agentadministered to a subject (e.g., a mammal, such as a human) may rangefrom about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50mg/kg of body weight. For example, the dose of sclerostin inhibitor(e.g., sclerostin binding agent) may range from about 1 mg/kg, about 5mg/kg, about 10 mg/kg, about 20 mg/kg, about 25 mg/kg, about 26 mg/kg,about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, about 30 mg/kg, about 31mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg,about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg, about 40mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44 mg/kg,about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, about 49mg/kg, or about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg,about 90 mg/kg, or about 95 mg/kg, up to about 100 mg/kg of body weight.In addition, it may be advantageous to administer multiple doses of asclerostin binding agent or space out the administration of doses,depending on the therapeutic regimen selected for a particular patient.For example, a dose of sclerostin inhibitor can be administered onceevery two weeks, once a week, twice a week, three times a week, fourtimes a week, or more, depending on the severity of the defect, the ageand physical health of the patient, and the like.

In some embodiments, the subject with the gap defect is optionallysuffering from a bone-related disorder selected from the groupconsisting of achondroplasia, cleidocranial dysostosis,enchondromatosis, fibrous dysplasia, Gaucher's Disease, hypophosphatemicrickets, Marfan's syndrome, multiple hereditary exotoses,neurofibromatosis, osteogenesis imperfecta, osteopetrosis,osteopoikilosis, sclerotic lesions, pseudoarthrosis, pyogenicosteomyelitis, periodontal disease, anti-epileptic drug induced boneloss, primary and secondary hyperparathyroidism, familialhyperparathyroidism syndromes, weightlessness induced bone loss,osteoporosis in men, postmenopausal bone loss, osteoarthritis, renalosteodystrophy, infiltrative disorders of bone, oral bone loss,osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis,metabolic bone diseases, mastocytosis, sickle cell anemia/disease, organtransplant related bone loss, kidney transplant related bone loss,systemic lupus erythematosus, ankylosing spondylitis, epilepsy, juvenilearthritides, thalassemia, mucopolysaccharidoses, Fabry Disease, TurnerSyndrome, Down Syndrome, Klinefelter Syndrome, leprosy, Perthe'sDisease, adolescent idiopathic scoliosis, infantile onset multi-systeminflammatory disease, Winchester Syndrome, Menkes Disease, Wilson'sDisease, ischemic bone disease (such as Legg-Calve-Perthes disease andregional migratory osteoporosis), anemic states, conditions caused bysteroids, glucocorticoid-induced bone loss, heparin-induced bone loss,bone marrow disorders, scurvy, malnutrition, calcium deficiency,osteoporosis, osteopenia, alcoholism, chronic liver disease,postmenopausal state, chronic inflammatory conditions, rheumatoidarthritis, inflammatory bowel disease, ulcerative colitis, inflammatorycolitis, Crohn's disease, oligomenorrhea, amenorrhea, pregnancy,diabetes mellitus, hyperthyroidism, thyroid disorders, parathyroiddisorders, Cushing's disease, acromegaly, hypogonadism, immobilizationor disuse, reflex sympathetic dystrophy syndrome, regional osteoporosis,osteomalacia, bone loss associated with joint replacement, HIVassociated bone loss, bone loss associated with loss of growth hormone,bone loss associated with cystic fibrosis, chemotherapy-associated boneloss, tumor-induced bone loss, cancer-related bone loss, hormoneablative bone loss, multiple myeloma, drug-induced bone loss, anorexianervosa, disease-associated facial bone loss, disease-associated cranialbone loss, disease-associated bone loss of the jaw, disease-associatedbone loss of the skull, bone loss associated with aging, facial boneloss associated with aging, cranial bone loss associated with aging, jawbone loss associated with aging, skull bone loss associated with aging,and bone loss associated with space travel.

In some embodiments, the subject is optionally suffering from (or hassuffered from) a cancer. The term “cancer” refers to a proliferativedisorder associated with uncontrolled cell proliferation, unrestrainedcell growth, and decreased cell death/apoptosis. Cancer includes, but isnot limited to, breast cancer, prostate cancer, lung cancer, kidneycancer, thyroid cancer, melanoma, follicular lymphomas, carcinomas withp53 mutations, and hormone-dependent tumors, including, but not limitedto, colon cancer, cardiac tumors, pancreatic cancer, retinoblastoma,glioblastoma, intestinal cancer, testicular cancer, stomach cancer,neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma,osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, Kaposi's sarcoma,ovarian cancer, leukemia (including acute leukemias (for example, acutelymphocytic leukemia, acute myelocytic leukemia, including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (for example, chronic myelocytic (granulocytic)leukemia and chronic lymphocytic leukemia), myelodysplastic syndromepolycythemia vera, lymphomas (for example, Hodgkin's disease andnon-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain diseases, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilm's tumor, cervical cancer, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, and menangioma.The terms “metastasis” and “cancer metastasis” are used interchangeablyherein to refer to the ability of a cancer cell to spread to othertissues. For example, “metastasis to bone” refers to the ability ofcertain types of cancer including, but not limited to, breast, prostate,lung, kidney, thyroid, and melanoma, to metastasize to bone.

In some embodiments, the subject optionally suffers from an osteolyticdisorder. The term “osteolytic disorder” as used herein refers to anycondition that is caused by an increase in the activity of osteoclasts,which are cells responsible for bone resorption. The terms “osteolysis”and “osteolytic bone loss” are used interchangeably to refer toosteoclast-mediated bone resorption or bone loss associated with anosteolytic disorder. Osteolytic disorders occur in subjects with apredisposition to develop an osteolytic disorder, or they occur insubjects with a disease that leads to or contributes to an osteolyticdisorder by stimulating osteoclast activity. In some embodiments, theosteolytic disorder is osteolytic bone loss. In other embodiments, theosteolytic disorder is cancer metastasis-induced osteolytic bone loss.In further embodiments, the osteolytic bone disorder is a metabolic bonedisease, including but not limited to, endocrinopathies (e.g.,hypercortisolism, hypogonadism, primary or secondaryhyperparathyroidism, and hyperthyroidism); dietary deficiency, includingbut not limited to, rickets, osteomalacia, scurvy, and malnutrition;osteoporosis; drug use, including glucocorticoids(glucocorticoid-induced osteoperosis), heparin, and alcohol; chronicdisease, including malabsorption syndromes; chronic renal failure,including renal osteodystrophy; chronic liver disease, including hepaticosteodystrophy; inherited disease, including osteogenesis imperfecta andhomocystinuria; and bone inflammation associated with arthritis,rheumatoid arthritis, psoriatic arthritis, fibrous dysplasia,periodontal disease, and Paget's disease.

The terms “metastasis-induced osteolytic bone loss,” and “cancermetastasis-induced osteolytic bone loss,” are used interchangeablyherein to refer to osteolysis or osteolytic bone loss resulting fromcancer cell metastasis to bone. The term “cancer metastasis-inducedosteoclast activation” is used herein to refer to the ability of cancercells that have metastasized to bone to induce the activation ofosteoclasts.

The sclerostin inhibitor is preferably administered to a subject in aphysiologically-acceptable (e.g., pharmaceutical) composition, which caninclude carriers, excipients, or diluents. It will be appreciated thatthe sclerostin inhibitors (e.g., anti-sclerostin antibody) describedherein may be used in the preparation of a medicament for administrationusing any of the dosage and timing regimens disclosed herein.Pharmaceutical compositions and methods of treatment are disclosed inU.S. Patent Publication No. 20050106683, which is incorporated byreference herein. “Physiologically-acceptable” refers to molecularentities and compositions that do not produce an allergic or similaruntoward reaction when administered to a human. In addition, thecomposition administered to a subject may contain more than onesclerostin inhibitor (e.g., two anti-sclerostin antibodies, or asclerostin binding agent and a synthetic chemical sclerostin inhibitor)or a sclerostin inhibitor in combination with one or more therapeuticshaving different mechanisms of action.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., subcutaneous, oral, parenteral, intravenous,intranasal, and intramuscular administration and formulation, is wellknown in the art and discussed in U.S. Patent Publication No.20070110747. For example, in certain circumstances, it will be desirableto deliver a pharmaceutical composition comprising a sclerostin bindingagent subcutaneously, parenterally, intravenously, intramuscularly, oreven intraperitoneally. Such approaches are well known to the skilledartisan, some of which are further described, for example, in U.S. Pat.Nos. 5,543,158; 5,641,515; and 5,399,363. Illustrative pharmaceuticalforms suitable for injectable use include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions (for example, see U.S. Pat.No. 5,466,468). In all cases the form must be sterile and must be fluidto the extent that easy syringability exists.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, for example, Remington's PharmaceuticalSciences, 15th ed., Mack Pub. Co., Easton, Pa., pp. 1035-1038 and1570-1580). Some variation in dosage and frequency of administration mayoccur depending on the condition of the subject being treated; age,height, weight, and overall health of the patient; and the existence ofany side effects. In addition, a pharmaceutical composition comprising asclerostin binding agent may be placed within containers (e.g. vials),along with packaging material that provides instructions regarding theuse of such pharmaceutical compositions. Generally, such instructionswill include a tangible expression describing the reagent concentration,as well as within certain embodiments, relative amounts of excipientingredients or diluents (e.g., water, saline or PBS) that may benecessary to reconstitute the pharmaceutical composition.

The methods described herein comprise administering an amount of a“sclerostin inhibitor.” As used herein, the term “sclerostin inhibitor”means any molecule that inhibits the biological activity of sclerostinon bone, as measured by changes to bone mineralization, bone density,effect on osteoblasts and/or osteoclasts, markers of bone formation,markers of bone resorption, markers of osteoblast activity, and/ormarkers of osteoclast activity. Such inhibitors may act by binding tosclerostin or its receptor or binding partner. Inhibitors in thiscategory include “sclerostin binding agents,” such as, e.g., antibodiesor peptide-based molecules. “Sclerostin inhibitors” also refers to smallorganic chemical compounds, optionally of less than about 1000 Daltonsin molecular weight that bind sclerostin and inhibit its activity.Inhibitors may alternatively act by inhibiting expression of sclerostin.Inhibitors in this category include polynucleotides or oligonucleotidesthat bind to sclerostin DNA or mRNA and inhibit sclerostin expression,including an antisense oligonucleotide, inhibitory RNA, DNA enzyme,ribozyme, an aptamer or pharmaceutically acceptable salts thereof thatinhibit the expression of sclerostin.

A “sclerostin binding agent” specifically binds to sclerostin orportions thereof to block or impair binding of human sclerostin to oneor more ligands. Sclerostin, the product of the SOST gene, is absent insclerosteosis, a skeletal disease characterized by bone overgrowth andstrong dense bones (Brunkow et al., Am. J. Hum. Genet., 68:577-589(2001); Balemans et al., Hum. Mol. Genet., 10:537-543 (2001)). The aminoacid sequence of human sclerostin is reported by Brunkow et al. and isdisclosed in U.S. Patent Publication No. 20070110747 as SEQ ID NO: 1(which patent publication is incorporated in its entirety for itsdescription of sclerostin binding agents and Sequence Listing).Recombinant human sclerostin/SOST is commercially available from R&DSystems (Minneapolis, Minn., USA; 2006 Catalog #1406-ST-025).Additionally, recombinant mouse sclerostin/SOST is commerciallyavailable from R&D Systems (Minneapolis, Minn., USA; 2006 Catalog#1589-ST-025). Research grade sclerostin-binding monoclonal antibodiesare commercially available from R&D Systems (Minneapolis, Minn., USA;mouse monoclonal: 2006 Catalog # MAB1406; rat monoclonal: 2006 Catalog #MAB1589). U.S. Pat. Nos. 6,395,511 and 6,803,453, and U.S. PatentPublication Nos. 20040009535 and 20050106683 refer to anti-sclerostinantibodies generally. Examples of sclerostin binding agents suitable foruse in the context of the invention also are described in U.S. PatentPublication Nos. 20070110747 and 20070072797, which are herebyincorporated by reference. Additional information regarding materialsand methods for generating sclerostin binding agents can be found inU.S. Patent Publication No. 20040158045 (hereby incorporated byreference).

The sclerostin binding agent of the invention preferably is an antibody.The term “antibody” refers to an intact antibody, or a binding fragmentthereof. An antibody may comprise a complete antibody (immunoglobulin)molecule (including polyclonal, monoclonal, chimeric, humanized, and/orhuman versions having full length heavy and/or light chains), orcomprise an antigen binding fragment thereof. Antibody fragments includeF(ab′)₂, Fab, Fab′, Fv, Fc, and Fd fragments, and can be incorporatedinto single domain antibodies (e.g., nanobodies), single-chainantibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, NatureBiotechnology, 23(9):1126-1136 (2005)). Antibody polypeptides, includingfibronectin polypeptide monobodies, also are disclosed in U.S. Pat. No.6,703,199. Other antibody polypeptides are disclosed in U.S. PatentPublication No. 20050238646. U.S. Pat. Nos. 6,395,511 and 6,803,453, andU.S. Patent Publication Nos. 20040009535 and 20050106683 (incorporatedin their entirety by reference for their disclosure of anti-sclerostinantibodies) refer to anti-sclerostin antibodies generally. The aminoacid sequence of human sclerostin is set forth in SEQ ID NO: 1 of theSequence Listing and is provided as SEQ ID NO: 1 of U.S. PatentPublication No. 20070110747 (which patent publication is incorporated inits entirety for its description of sclerostin and sclerostin bindingagents and Sequence Listing). Sclerostin also is described in Brunkow etal., Am. J. Hum. Genet., 68:577-589 (2001); and Balemans et al., Hum.Mol. Genet., 10:537-543 (2001). Additional information regardingmaterials and methods for generating anti-sclerostin antibodies can befound in U.S. Patent Publication No. 20040158045 (hereby incorporated byreference in its entirety).

An antibody fragment may be any synthetic or genetically engineeredprotein. For example, antibody fragments include isolated fragmentsconsisting of the light chain variable region, “Fv” fragments consistingof the variable regions of the heavy and light chains, and recombinantsingle chain polypeptide molecules in which light and heavy variableregions are connected by a peptide linker (scFv proteins).

Another form of an antibody fragment is a peptide comprising one or morecomplementarity determining regions (CDRs) of an antibody. CDRs (alsotermed “minimal recognition units” or “hypervariable region”) can beobtained by constructing polynucleotides that encode the CDR ofinterest. Such polynucleotides are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region using mRNAof antibody-producing cells as a template (see, for example, Larrick etal., Methods: A Companion to Methods in Enzymology, 2:106 (1991);Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies Production, Engineering and Clinical Application,Ritter et al. (eds.), page 166, Cambridge University Press (1995); andWard et al., “Genetic Manipulation and Expression of Antibodies,” inMonoclonal Antibodies: Principles and Applications, Birch et al.,(eds.), page 137, Wiley-Liss, Inc. (1995)).

Anti-sclerostin antibodies may bind to sclerostin of SEQ ID NO: 1, or anaturally occurring variant thereof, with an affinity (Kd) of less thanor equal to 1×10⁻⁷M, less than or equal to 1×10⁻⁸M, less than or equalto 1×10⁻⁹M, less than or equal to 1×10⁻¹⁰ M, less than or equal to1×10⁻¹¹M, or less than or equal to 1×10⁻¹²M. Affinity is determinedusing a variety of techniques, an example of which is an affinity ELISAassay. In various embodiments, affinity is determined by a BIAcoreassay. In various embodiments, affinity is determined by a kineticmethod. In various embodiments, affinity is determined by anequilibrium/solution method. U.S. Patent Publication No. 20070110747contains additional description of affinity assays suitable fordetermining the affinity (Kd) of an antibody for sclerostin.

Anti-sclerostin antibodies for use in the methods described hereinpreferably modulate sclerostin function in the cell-based assaydescribed in U.S. Patent Publication No. 20070110747 and/or the in vivoassay described in U.S. Patent Publication No. 20070110747 and/or bindto one or more of the epitopes described in U.S. Patent Publication No.20070110747 and/or cross-block the binding of one of the antibodiesdescribed in U.S. Patent Publication No. 20070110747 and/or arecross-blocked from binding sclerostin by one of the antibodies describedin U.S. Patent Publication No. 20070110747 (incorporated by reference inits entirety and for its description of assays for characterizing ananti-sclerostin antibody).

In some or any embodiments, the anti-sclerostin antibody binds to asclerostin polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 1 and binds the sequence of SEQ ID NO: 6(CGPARLLPNAIGRGKWWRPSGPDFRC; corresponding to amino acids 86-111 of SEQID NO: 1). Alternatively or in addition, the anti-sclerostin antibodybinds to a sclerostin polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 1 and binds the sequence of at least one of SEQ IDNO: 2 (DVSEYSCRELHFTR; corresponding to amino acids 51-64 of SEQ ID NO:1), SEQ ID NO: 3 (SAKPVTELVCSGQCGPAR; corresponding to amino acids 73-90of SEQ ID NO: 1), SEQ ID NO: 4 (WWRPSGPDFRCIPDRYR; corresponding toamino acids 101-117 of SEQ ID NO: 1), SEQ ID NO: 5 (LVASCKCKRLTR;corresponding to amino acids 138-149 of SEQ ID NO: 1), SEQ ID NO: 70(SAKPVTELVCSGQC; corresponding to amino acids 73-86 of SEQ ID NO: 1),SEQ ID NO: 71 (LVASCKC; corresponding to amino acids 138-144 of SEQ IDNO: 1), SEQ ID NO: 72 (C1RELHFTR; corresponding to amino acids 57-64 ofSEQ ID NO: 1), or SEQ ID NO: 73 (CIPDRYR; corresponding to amino acids111-117 of SEQ ID NO: 1) within SEQ ID NO: 1. For example, in oneaspect, the anti-sclerostin antibody binds a subregion of sclerostin ofSEQ ID NO: 1 comprising SEQ ID NOs: 2-5 (and/or SEQ ID NOs: 70-73),optionally in its native three-dimensional conformation. Optionally, theanti-sclerostin antibody binds a peptide consisting of one or more ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73 (e.g., apeptide consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQID NO: 5 or a peptide consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ IDNO: 72, and SEQ ID NO: 73).

In some or any embodiments, the anti-sclerostin antibody binds to asclerostin polypeptide having the amino acid sequences of SEQ ID NO:2,SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, wherein SEQ ID NO:2 and 4 arejoined by a disulfide bond at amino acid positions 57 and 111 withreference to SEQ ID NO:1, and SEQ ID NO:3 and 5 are joined by at leastone of (a) a disulfide bond at amino acid positions 82 and 142 withreference to SEQ ID NO:1, and (b) a disulfide bond at amino acidpositions 86 and 144 with reference to SEQ ID NO:1; the polypeptide mayretain the tertiary structure of the corresponding polypeptide region ofhuman sclerostin of SEQ ID NO:1. Alternatively or in addition, thesclerostin binding agent (e.g., anti-sclerostin antibody) binds apolypeptide having the amino acid sequences of SEQ ID NO: 70, SEQ ID NO:71, SEQ ID NO: 72 and SEQ ID NO: 73, wherein SEQ ID NO: 72 and 73 arejoined by a disulfide bond at amino acid positions 57 and 111 withreference to SEQ ID NO: 1, and SEQ ID NO: 70 and 71 are joined by atleast one of (a) a disulfide bond at amino acid positions 82 and 142with reference to SEQ ID NO: 1, and (b) a disulfide bond at amino acidpositions 86 and 144 with reference to SEQ ID NO: 1.

In various aspects, the anti-sclerostin antibody is capable ofneutralizing human sclerostin in a MC3T3 cell-based mineralization assaywhen there is less than a 6-fold excess of moles of sclerostin bindingsites per well as compared to the number of moles of sclerostin perwell. Mineralization by osteoblast-lineage cells in culture, eitherprimary cells or cell lines, is used as an in vitro model of boneformation. An exemplary cell-based mineralization assay is described inU.S. Patent Publication No. 20070110747 at, e.g., Example 8 (herebyincorporated by reference). MC3T3-E1 cells (Sudo et al., J. Cell Biol.,96:191-198 (1983)) and subclones of the original cell line can formmineral in culture upon growth in the presence of differentiatingagents. Such subclones include MC3T3-E1-BF (Smith et al., J. Biol.Chem., 275:19992-20001 (2000)). For both the MC3T3-E1-BF subclone aswell as the original MC3T3-E1 cells, sclerostin can inhibit one or moreof the sequence of events leading up to and including mineral deposition(i.e., sclerostin inhibits mineralization). Anti-sclerostin antibodiesthat are able to neutralize sclerostin's inhibitory activity allow formineralization of the culture in the presence of sclerostin such thatthere is a statistically significant increase in, e.g., deposition ofcalcium phosphate (measured as calcium) as compared to the amount ofcalcium measured in the sclerostin-only (i.e., no antibody) treatmentgroup.

When running the assay with the goal of determining whether a particularanti-sclerostin antibody can neutralize sclerostin, the amount ofsclerostin used in the assay desirably is the minimum amount ofsclerostin that causes at least a 70%, statistically significant,reduction in deposition of calcium phosphate (measured as calcium) inthe sclerostin-only group, as compared to the amount of calcium measuredin the no sclerostin group. An anti-sclerostin neutralizing antibody isdefined as one that causes a statistically significant increase indeposition of calcium phosphate (measured as calcium) as compared to theamount of calcium measured in the sclerostin-only (i.e., no antibody)treatment group. To determine whether an anti-sclerostin antibody isneutralizing or not, the amount of anti-sclerostin antibody used in theassay needs to be such that there is an excess of moles of sclerostinbinding sites per well as compared to the number of moles of sclerostinper well. Depending on the potency of the antibody, the fold excess thatmay be required can be 24, 18, 12, 6, 3, or 1.5, and one of skill isfamiliar with the routine practice of testing more than oneconcentration of binding agent (antibody). For example, a very potentanti-sclerostin neutralizing antibody will neutralize sclerostin whenthere is less than a 6-fold excess of moles of sclerostin binding sitesper well as compared to the number of moles of sclerostin per well. Aless potent anti-sclerostin neutralizing antibody will neutralizesclerostin only at a 12, 18 or 24 fold excess.

The anti-sclerostin antibody optionally has an IC₅₀ of 100 nM or less,or 75 nM or less, or 50 nM or less, or 25 nM or less for neutralizinghuman sclerostin in a cell-based assay, such as a bone specific alkalinephosphatase assay, e.g., the bone specific alkaline phosphatase assaydescribed in International Patent Publication No. WO 2008/115732 andU.S. Pat. No. 7,744,874 (incorporated herein by reference in itsentirety for its description of cell-based assays and anti-sclerostinantibodies). The bone specific alkaline phosphatase assay is predicatedon the ability of sclerostin to decrease BMP-4 and Wnt3a-stimulatedalkaline phosphatase levels in the multipotential murine cell line,C2C12. According to WO 2008/115732, a neutralizing anti-sclerostinantibody mediates a dose-dependent increase of alkaline phosphataseactivity in this assay. Exemplary protocols of the cell-based assays areprovided in Example 1.

Alternatively or in addition, the anti-sclerostin antibody has an IC₅₀of 100 nM or less (e.g., 75 nM or less, or 50 nM or less) forneutralizing human sclerostin in a cell-based Wnt signaling assay inHEK293 cell lines, such as the Wnt assay involving Wnt1-mediatedinduction of STF reporter gene described in e.g., International PatentPublication No. WO 2009/047356 (incorporated by reference for itsdiscussion of anti-sclerostin antibodies and cell-based assays).Alternatively or in addition, the anti-sclerostin antibody has an IC₅₀of 500 nM or less (e.g., 250 nM or less, 150 nM or less, 100 nM or less,or 50 nM or less) for neutralizing human sclerostin in a BMP2-inducedmineralization assay in MC3T3 cells, such as the mineralization assaydescribed in e.g., International Patent Publication No. WO 2009/047356.An exemplary protocol is provided in Example 1.

Examples of anti-sclerostin antibodies suitable for use in the contextof the invention are described in U.S. Patent Publication Nos.20070110747 and 20070072797, which are hereby incorporated by reference.In one embodiment of the invention, the anti-sclerostin antibodycross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C,Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10,Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20,Ab-21, Ab-22, Ab-23, and Ab-24 (all of which are described in U.S.Patent Publication No. 20070110747) to sclerostin. Alternatively or inaddition, the anti-sclerostin antibody is cross-blocked from binding tosclerostin by at least one of antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1,Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12,Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22,Ab-23, and Ab-24 (all of which are described in U.S. Patent PublicationNo. 20070110747). The terms “cross-block,” “cross-blocked,” and“cross-blocking” are used interchangeably herein to mean the ability ofan antibody to interfere with the binding of other antibodies tosclerostin. The extent to which an antibody is able to interfere withthe binding of another to sclerostin, and therefore whether it can besaid to cross-block, can be determined using competition binding assays.In some aspects of the invention, a cross-blocking antibody or fragmentthereof reduces sclerostin binding of a reference antibody between about40% and about 100%, such as about 60% and about 100%, specificallybetween 70% and 100%, and more specifically between 80% and 100%. Aparticularly suitable quantitative assay for detecting cross-blockinguses a Biacore machine which measures the extent of interactions usingsurface plasmon resonance technology. Another suitable quantitativecross-blocking assay uses an ELISA-based approach to measure competitionbetween antibodies in terms of their binding to sclerostin.

Examples of suitable anti-sclerostin antibodies and fragments thereofinclude antibodies and antibody fragments having one or more of CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 specifically disclosed in U.S.Patent Publication No. 20070110747. At least one of the regions ofCDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 may have at least oneamino acid substitution, provided that the antibody retains the bindingspecificity of the non-substituted CDR. Preferably, the anti-sclerostinantibody is Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6,Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16,Ab-17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, or Ab-24 of U.S. PatentPublication No. 20070110747.

In addition, the anti-sclerostin antibody can comprise at least one CDRsequence having at least 75% identity (e.g., 100% identity) to a CDRselected from SEQ ID NOs: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 78, 79, 80, 81, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 351, 352, 353, 358, 359,and 360 provided in the Sequence Listing and disclosed in U.S. PatentPublication No. 20070110747. Preferably, the anti-sclerostin antibodycomprises at least one CDR sequence having at least 75% identity to aCDR selected from SEQ ID NOs: 245, 246, 247, 78, 79, 80, 269, 270, 271,239, 240, and 241, all of which is provided in the Sequence Listing anddescribed in U.S. Patent Publication No. 20070110747. As described inU.S. Patent Publication No. 20070110747, the anti-sclerostin antibodycan comprise: a) CDR sequences of SEQ ID NOs:54, 55, and 56 and CDRsequences of SEQ ID NOs:51, 52, and 53; b) CDR sequences of SEQ IDNOs:60, 61, and 62 and CDR sequences of SEQ ID NOs:57, 58, and 59; c)CDR sequences of SEQ ID NOs:48, 49, and 50 and CDR sequences of SEQ IDNOs:45, 46, and 47; d) CDR sequences of SEQ ID NOs:42, 43, and 44 andCDR sequences of SEQ ID NOs:39, 40, and 41; e) CDR sequences of SEQ IDNOs:275, 276, and 277 and CDR sequences of SEQ ID NOs:287, 288, and 289;f) CDR sequences of SEQ ID NOs:278, 279, and 280 and CDR sequences ofSEQ ID NOs:290, 291, and 292; g) CDR sequences of SEQ ID NOs:78, 79, and80 and CDR sequences of SEQ ID NOs: 245, 246, and 247; h) CDR sequencesof SEQ ID NOs:81, 99, and 100 and CDR sequences of SEQ ID NOs:248, 249,and 250; i) CDR sequences of SEQ ID NOs:101, 102, and 103 and CDRsequences of SEQ ID NOs:251, 252, and 253; j) CDR sequences of SEQ IDNOs:104, 105, and 106 and CDR sequences of SEQ ID NOs:254, 255, and 256;k) CDR sequences of SEQ ID NOs:107, 108, and 109 and CDR sequences ofSEQ ID NOs:257, 258, and 259; 1) CDR sequences of SEQ ID NOs:110, 111,and 112 and CDR sequences of SEQ ID NOs:260, 261, and 262; m) CDRsequences of SEQ ID NOs:281, 282, and 283 and CDR sequences of SEQ IDNOs:293, 294, and 295; n) CDR sequences of SEQ ID NOs:113, 114, and 115and CDR sequences of SEQ ID NOs:263, 264, and 265; o) CDR sequences ofSEQ ID NOs:284, 285, and 286 and CDR sequences of SEQ ID NOs:296, 297,and 298; p) CDR sequences of SEQ ID NOs:116, 237, and 238 and CDRsequences of SEQ ID NOs:266, 267, and 268; q) CDR sequences of SEQ IDNOs:239, 240, and 241 and CDR sequences of SEQ ID NOs:269, 270, and 271;r) CDR sequences of SEQ ID NOs:242, 243, and 244 and CDR sequences ofSEQ ID NOs:272, 273, and 274; or s) CDR sequences of SEQ ID NOs:351,352, and 353 and CDR sequences of SEQ ID NOs:358, 359, and 360.

The anti-sclerostin antibody also can comprise at least one CDR sequencehaving at least 75% identity (e.g., 100% identical) to a CDR selectedfrom CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 wherein CDR-H1has the sequence given in SEQ ID NO: 245, CDR-H2 has the sequence givenin SEQ ID NO: 246, CDR-H3 has the sequence given in SEQ ID NO: 247,CDR-L1 has the sequence given in SEQ ID NO: 78, CDR-L2 has the sequencegiven in SEQ ID NO: 79 and CDR-L3 has the sequence given in SEQ ID NO:80, all of which is provided in the Sequence Listing and described inU.S. Patent Publication No. 20070110747. The anti-sclerostin antibody,in various aspects, comprises two of the CDRs or six of the CDRs.Optionally, the anti-sclerostin antibody comprises heavy chainscomprising SEQ ID NO: 378 and light chains comprising SEQ ID NO 376.

The anti-sclerostin antibody also can comprise at least one CDR sequencehaving at least 75% identity (e.g., 100% identical) to a CDR selectedfrom CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 wherein CDR-H1has the sequence given in SEQ ID NO: 269, CDR-H2 has the sequence givenin SEQ ID NO: 270, CDR-H3 has the sequence given in SEQ ID NO: 271,CDR-L1 has the sequence given in SEQ ID NO: 239, CDR-L2 has the sequencegiven in SEQ ID NO: 240 and CDR-L3 has the sequence given in SEQ ID NO241, all of which is provided in the Sequence Listing and described inU.S. Patent Publication No. 20070110747. The anti-sclerostin antibody,in various aspects, comprises at least two of the CDRs or six of theCDRs.

Alternatively, the anti-sclerostin antibody can have a heavy chaincomprising CDR's H1, H2, and H3 and comprising a polypeptide having thesequence provided in SEQ ID NO: 137 or a variant thereof in which saidCDR's are at least 75% identical (e.g., 100% identical) to SEQ ID NO:245, 246, and 247, respectively, and a light chain comprising CDR's L1,L2 and L3 and comprising a polypeptide having the sequence provided inSEQ ID NO: 133 or a variant thereof in which said CDR's are at least 75%identical (e.g., 100% identical) to SEQ ID NO: 78, 79, and 80,respectively (as described in U.S. Patent Publication No. 20070110747).

The anti-sclerostin antibody may have a heavy chain comprising CDR's H1,H2, and H3 and comprising a polypeptide having the sequence provided inSEQ ID NO: 145 or 392 or a variant thereof in which said CDR's are atleast 75% identical (e.g., 100% identical) to SEQ ID NO: 245, 246, and247, respectively, and a light chain comprising CDR's L1, L2, and L3 andcomprising a polypeptide having the sequence provided in SEQ ID NO: 141or a variant thereof in which said CDR's are at least 75% identical(e.g., 100% identical) to SEQ ID NO: 78, 79, and 80, respectively (asdescribed in U.S. Patent Publication No. 20070110747).

The anti-sclerostin antibody may have a heavy chain comprising CDR's H1,H2, and H3 and comprising a polypeptide having the sequence provided inSEQ ID NO: 335, 331, 345, or 396 or a variant of any of the foregoing inwhich said CDR's are at least 75% (e.g., 100% identical) identical toSEQ ID NO: 269, 270, and 271, respectively, and a light chain comprisingCDR's L1, L2, and L3 and comprising a polypeptide having the sequenceprovided in SEQ ID NO: 334 or 341 or a variant of any of the foregoingin which said CDR's are at least 75% identical (e.g., 100% identical) toSEQ ID NO: 239, 240, and 241, respectively (as described in U.S. PatentPublication No. 20070110747). All combinations of the heavy and lightchain sequences are contemplated (e.g., heavy chains comprising SEQ IDNO: 335 and light chains comprising SEQ ID NO: 334; heavy chainscomprising SEQ ID NO: 331 and light chains comprising SEQ ID NO: 334 or341; and heavy chains comprising SEQ ID NO: 345 or 396 and light chainscomprising SEQ ID NO: 341).

Alternatively, the anti-sclerostin antibody has a heavy chain comprisinga polypeptide having the sequence provided in SEQ ID NO:137, and a lightchain comprising a polypeptide having the sequence provided in SEQ IDNO:133; a heavy chain comprising a polypeptide having the sequenceprovided in SEQ ID NO:145 or 392, and a light chain comprising apolypeptide having the sequence provided in SEQ ID NO: 141; a heavychain comprising a polypeptide having the sequence provided in SEQ IDNO:335, and a light chain comprising a polypeptide having the sequenceprovided in SEQ ID NO:334; a heavy chain comprising a polypeptide havingthe sequence provided in SEQ ID NO:331, and a light chain comprising apolypeptide having the sequence provided in SEQ ID NO:341; or a heavychain comprising a polypeptide having the sequence provided in SEQ IDNO:345 or 396, and a light chain comprising a polypeptide having thesequence provided in SEQ ID NO:341 (as described in U.S. PatentPublication No. 20070110747).

Examples of anti-sclerostin antibodies also include, but are not limitedto, the anti-sclerostin antibodies disclosed in International PatentPublication Nos. WO 2008/092894, WO 2008/115732, WO 2009/056634, WO2009/047356, WO 2010/100200, WO 2010/100179, WO 2010/115932, and WO2010/130830 (each of which is incorporated by reference herein in itsentirety), such as an anti-sclerostin antibody comprising CDRs of SEQ IDNOs: 20-25 of International Patent Publication No. WO 2008/115732 (SEQID NOs: 416-421 herein), an anti-sclerostin antibody comprising CDRs ofSEQ ID NOs: 26-31 of International Patent Publication No. WO 2008/115732(SEQ ID NOs: 422-427 herein), an anti-sclerostin antibody comprisingCDRs of SEQ ID NOs: 32-37 of International Patent Publication No. WO2008/115732 (SEQ ID NOs: 428-433 herein), an anti-sclerostin antibodycomprising CDRs of SEQ ID NOs: 4, 15, 26, 37, 48, and 59 ofInternational Patent Publication No. WO 2009/047356 (SEQ ID NOs: 443,454, 465, 476, 487 and 498, respectively, herein), or an anti-sclerostinantibody comprising the amino acid sequence of at least one of SEQ IDNOs: 135-143, 153-161, or 171-179 of International Patent PublicationNo. WO 2010/130830 (SEQ ID NOs: 745-753, 763-771, 781-789, respectively,herein).

Alternatively, the methods described herein comprise administering asclerostin inhibitor other than an anti-sclerostin antibody. Such agentscan act directly or indirectly on SOST or sclerostin. Sclerostininhibitors contemplated for use in the methods described herein includethose described in U.S. Patent Publication No. 20030229041 (the entiredisclosure of which is hereby incorporated by reference, with particularemphasis upon the description of sclerostin inhibitors). For example,agents useful for modulating SOST expression and sclerostin activityinclude, but are not limited to, steroids (such as those correspondingto Formula 1 of U.S. Patent Publication No. 20030229041), alkaloids,terpenoids, peptoids, and synthetic chemicals. In some embodiments, theSOST antagonist or agonist can bind to a glucocorticoid receptor. Forexample, dexamethasone tends to abolish the stimulatory effect of BMP-4and BMP-6 on SOST expression. Other chemical entities includingglucocorticoid analogs, bile salts (such as those corresponding toFormula 3 of U.S. Patent Publication No. 20030229041), andprostaglandins (such as those corresponding to Formula 2 of U.S. PatentPublication No. 20030229041) also modulate the effects of bonemorphogenetic proteins on SOST expression, and are contemplated for usein the methods described herein.

Sclerostin expression inhibitors that may be used according to themethods described herein include inhibitory nucleic acids, includingpharmaceutically acceptable salts thereof, e.g., sodium salts. In someembodiments, the inhibitory nucleic acid as described elsewhere hereinis selected from the group consisting of antisense oligonucleotides,ribozymes, external guide sequence (EGS) oligonucleotides, siRNAcompounds, single- or double-stranded RNA interference (RNAi) compoundssuch as siRNA compounds, modified bases/locked nucleic acids (LNAs),antagomirs, peptide nucleic acids (PNAs), and other oligomeric compoundsor oligonucleotide mimetics which hybridize to at least a portion of thetarget nucleic acid and modulate its function. In some embodiments, theinhibitor nucleic acid is single stranded or double stranded. In someembodiments, the inhibitory nucleic acid is an antisenseoligonucleotide, modified bases/locked nucleic acids (LNA), peptidenucleic acids (PNA), arabinonucleic acids (ANA) (as described, forexample, in PCT Publication No. WO 99/67378); 2′-fluoro-D-Arabinonucleicacids (FANA) (as described in, for example, Lon et al., Biochem.,41:3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett.,12:2651-2654, 2002; the disclosures of which are incorporated herein byreference in their entireties); phosphorodiamidate morpholino oligomers(PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238,2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosuresof which are incorporated herein by reference in their entireties);ethylene bridged nucleic acids (as described in, for example,International Patent Publication No. WO 2005/042777, Morita et al.,Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. GeneTher., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149,2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005;the disclosures of which are incorporated herein by reference in theirentireties); 2′-0,4′-C-ethylene-bridged nucleic acid, ribozyme, externalguide sequence (EGS) oligonucleotides, microRNAs (miRNAs), small,temporal RNAs (stRNAs), and single- or double-stranded RNA interference(RNAi) compounds or siRNA. In some embodiments, the inhibitory nucleicacid comprises at least one nucleotide and/or nucleoside modification(e.g., oligonucleotides with modified backbones or modified sugarmoieties).

Activity of a particular sclerostin inhibitor, e.g., anti-sclerostinantibody, for use in the methods described herein may be measured in avariety of ways, including the methods described above for detectingincreases in bone mineral content or bone density. The ability of asclerostin inhibitor to modulate bone mass may be calculated from bodyweights or by using other methods (see Guinness-Hey, Metab. Bone Dis.Relat. Res., 5:177-181 (1984)). Animals and particular animal models areused in the art for testing the effect of the pharmaceuticalcompositions and methods on, for example, parameters of bone loss, boneresorption, bone formation, bone strength, or bone mineralization.Examples of such models include the ovariectomized rat model (Kalu, Boneand Mineral, 15:175-192 (1991); Frost and Jee, Bone and Mineral,18:227-236 (1992); and Jee and Yao, J. Musculoskel. Neuron. Interact.,1:193-207 (2001)). The methods for measuring sclerostin binding agentactivity described herein also may be used to determine the efficacy ofother sclerostin inhibitors.

Alternatively, a sclerostin inhibitor can be selected based on itsability to modulate bone marker levels. Bone markers are productscreated during the bone remodeling process and are released by bone,osteoblasts, and/or osteoclasts. Fluctuations in bone resorption and/orbone formation “marker” levels imply changes in boneremodeling/modeling. The International Osteoporosis Foundation (IOF)recommends using bone markers to monitor bone density therapies (see,e.g., Delmas et al., Osteoporos Int., Suppl. 6:S2-17 (2000),incorporated herein by reference). Markers indicative of bone resorption(or osteoclast activity) include, for example, C-telopeptide (e.g.,C-terminal telopeptide of type 1 collagen (CTX) or serum cross-linkedC-telopeptide), N-telopeptide (N-terminal telopeptide of type 1 collagen(NTX)), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline,galactosyl hydroxylysine, and tartrate-resistant acid phosphatase (e.g.,serum tartrate-resistant acid phosphatase isoform 5b). Boneformation/mineralization markers include, but are not limited to,bone-specific alkaline phosphatase (BSAP), peptides released from N- andC-terminal extension of type I procollagen (P1NP, PICP), and osteocalcin(OstCa). Several kits are commercially-available to detect and quantifymarkers in clinical samples, such as urine and blood.

Various routes of administering a sclerostin inhibitor (e.g., ananti-sclerostin antibody) to a subject are known in the art anddiscussed in, e.g., U.S. Patent Publication No. 20070110747, thedisclosure of which is incorporated herein by reference in its entirety.For example, in various embodiments, it is desirable to deliver apharmaceutical composition comprising the anti-sclerostin antibodysubcutaneously, parenterally, intravenously, intramuscularly, or evenintraperitoneally. Such approaches are well known to the skilledartisan, some of which are further described, for example, in U.S. Pat.Nos. 5,543,158; 5,641,515; and 5,399,363. Illustrativephysiologically-acceptable (e.g., pharmaceutical) forms suitable for useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). The form must besterile and is desirably fluid to the extent that easy syringabilityexists (i.e., is not excessively viscous so as to prevent passagethrough a syringe). A pharmaceutical composition comprising theanti-sclerostin antibody may be placed within containers (e.g., vials orsyringes), along with packaging material that provides instructionsregarding the use of such pharmaceutical compositions. Generally, suchinstructions will include a tangible expression describing the reagentconcentration, as well as within certain embodiments, relative amountsof excipient ingredients or diluents (e.g., water, saline or PBS) thatmay be necessary to reconstitute the pharmaceutical composition.

Treatment of a pathology by combining two or more agents that target thesame pathogen or biochemical pathway sometimes results in greaterefficacy and diminished side effects relative to the use of thetherapeutically relevant dose of each agent alone. In some cases, theefficacy of the drug combination is additive (the efficacy of thecombination is approximately equal to the sum of the effects of eachdrug alone), but in other cases the effect can be synergistic (theefficacy of the combination is greater than the sum of the effects ofeach drug given alone). As used herein, the term “combination therapy”means the two compounds can be delivered in a simultaneous manner, e.g.concurrently, or wherein one of the compounds is administered first,followed by the second agent, e.g., sequentially. The desired result canbe either a subjective relief of one or more symptoms or an objectivelyidentifiable improvement in the recipient of the dosage.

In some embodiments, the sclerostin inhibitor (e.g. anti-sclerostinantibody) is administered in combination with the use of materials thatpromote the regrowth of bone such as bone graft, bone dust, bone chips,demineralized bone matrix, bone scaffolds, prosthesis, metalstabilizers, or bone scaffold substances comprising one or more ofpolymers, ceramics, cement and calcium phosphates-based bone-graftsubstitutes. Many variations of such materials are known in the art.

In some embodiments, the sclerostin inhibitor (e.g., anti-sclerostinantibody) is administered along with a second bone-enhancing therapeuticuseful for the treatment of decreased bone mineral density or bonedefect. In some embodiments, the bone-enhancing therapeutic is selectedfrom the group consisting of an anti-resorptive drug, a bone-formingagent, an estrogen receptor antagonist (including, but not limited to,raloxifene, bazedoxifene and lasofoxifene) and a drug that has aninhibitory effect on osteoclasts. In some embodiments, theanti-resorptive drug includes, but is not limited to, a bisphosphonate(including, but not limited to, alendronate, risedronate, ibandronateand zoledronate), an estrogen or estrogen analogue, an anti-RANK ligand(RANKL) antibody or RANKL inhibitor, vitamin D, or a vitamin Dderivative or mimic thereof, a selective estrogen receptor modulator(SERM) and a calcium source, Tibolone, calcitonin, a calcitriol andhormone replacement therapy. In some embodiments, the bone-enhancingagent includes, but is not limited to parathyroid hormone (PTH) or apeptide fragment thereof, PTH-related protein (PTHrp), bonemorphogenetic protein, osteogenin, NaF, a PGE₂ agonist, a statin,strontium ranelate, an anti-DKK1 antibody or inhibitor. In someembodiments, the bone-enhancing agent is Forteo® (Teriparatide),Preotact®, or Protelos®.

The invention is further described in the following examples. Thefollowing examples serve only to illustrate the invention and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1

This Example describes various cell-based neutralization assays usefulfor characterizing the neutralization activity of an anti-sclerostinantibody.

MC3T3 Cell-Based Mineralization Assay—

Ascorbic acid and B-glycerophosphate are used to induce MC3T3-E1-BF celldifferentiation leading to mineral deposition. An exemplary screeningprotocol, in 96-well format, involves plating cells on day 1, followedby seven media changes over a 12-day period with most of the mineraldeposition taking place in the final eighteen hours. The specifictiming, and extent, of mineral deposition may vary depending, in part,on the particular serum lot number being used. Control experiments willallow such variables to be accounted for, as is well known in the art ofcell culture experimentation generally. For statistical analysis (usingMS Excel and JMP) a 1-way-ANOVA followed by Dunnett's comparison may beused to determine differences between groups. Group means for each dataset are considered significantly different when the P value is less than0.05 (P<0.05).

Cell culture for expansion of MC3T3-E1-BF cells is performed as follows.Cell culture is performed at 37° C. and 5% CO₂. A cell bank can begenerated for the purposes of screening for sclerostin neutralizingantibodies. One vial of frozen MC3T3-E1-BF cells are thawed by agitationin a 37° C. water bath. The thawed cells are put into 10 mls ofExpansion Medium (Alpha-MEM/10% FBS/PenStrepGlu) in a 50 ml tube andgently spun down for 5 minutes. The cells are then resuspended in 4 mlsof Alpha-MEM/10% FBS/PenStrepGlu. After determining the number of cellsusing trypan blue and hemacytometer, 1×10⁶ cells are plated in 50 mlsAlpha-MEM/10% FBS/PenStrepGlu media in one T175 flask.

When this passage is confluent (at approximately 7 days), the cells aretrypsinized with trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spundown for 5 minutes and then resuspended in 5 mls Alpha-MEM/10%FBS/PenStrepGlu. After determining the number of cells using trypan blueand hemacytometer, cells are plated at 1×10⁶ cells in 50 mlsAlpha-MEM/10% FBS/PenStrepGlu media per one T175 flask. The number ofT175 flasks used for plating at this point depends upon the total cellnumber available and the desired number of flasks that are to be takenforward to the next passage.

When this passage is confluent (about 3-4 days), the cells aretrypsinized with trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spundown for 5 minutes and then resuspended in 5 mls Alpha-MEM/10%FBS/PenStrepGlu. After determining the number of cells using trypan blueand hemacytometer, cells are plated at 1×10⁶ cells in 50 mlsAlpha-MEM/10% FBS/PenStrepGlu media per one T175 flask. The number ofT175 flasks used for plating at this point depends upon the total cellnumber available and the desired number of flasks that were to be takenforward to the next passage.

When this passage is confluent (about 3-4 days), the cells aretrypsinized with trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spundown for 5 minutes and then resuspended in 5 mls Alpha-MEM/10%FBS/PenStrepGlu. After determining the number of cells using trypan blueand hemacytometer, cells are plated at 1×10⁶ cells in 50 mlsAlpha-MEM/10% FBS/PenStrepGlu media per one T175 flask. The number ofT175 flasks used for plating at this point depends upon the total cellnumber available and the desired number of flasks that were to be takenforward to the next passage. Extra cells are frozen down at 1-2×10⁶ livecells/ml in 90% FBS/10% DMSO.

When this passage is confluent (about 3-4 days), the cells weretrypsinized with trypsin/EDTA (0.05% Trypsin; 0.53 mM EDTA), gently spundown for 5 minutes and then resuspended in 5 mls Alpha-MEM/10%FBS/PenStrepGlu. After determining the number of cells using trypan blueand hemacytometer, the cells are frozen down at 1-2×10⁶ live cells/ml in90% FBS/10% DMSO. This “final passage” of frozen cells is the passageused for the screening assay.

Cell culture for mineralizing MC3T3-E1-BF cells is performed as follows.Cell culture is performed at 37° C. and 5% CO₂. It is desirable tominimize temperature and % CO₂ fluctuations during the mineralizationcell culture procedure. An appropriate number of “final passage” vialsprepared as described above are thawed by agitation in a 37° C. waterbath. The thawed cells are put into 10 mls of Expansion Medium(Alpha-MEM/10% FBS/PenStrepGlu) in a 50 ml tube and gently spun down for5 minutes. The cells are then resuspended in 4 mls of Alpha-MEM/10%FBS/PenStrepGlu. After determining the number of cells by trypan blueand hemacytometer, 2500 cells are plated in 200 microliters of Expansionmedia per well on collagen I coated 96-well plates (Becton DickinsonLabware, cat #354407).

An exemplary cell culture procedure is as follows. The starting day forplating the cells is indicated to be a Wednesday. If a different day ofthe week is used as the starting day for plating the cells, that daywill trigger the daily schedule for removing and adding media during theentire process as indicated below. For example, if the cells are platedon a Tuesday, media should not be removed and added on the first Fridayand Saturday, nor on the second Friday and Saturday. With a Tuesdaystart, the plates would be prepared for the calcium assay on the finalSunday. Cells are plated on a Wednesday at 2500 cells in 200 μl ofExpansion media. On Thursday all of the Expansion media is removed and200 μl of Differentiation Media is added. On Friday 100 μl of media isremoved and 100 μl of fresh Differentiation Media is added. On Monday100 μl of media is removed and 100 μl of fresh Differentiation Media isadded. On Tuesday 100 μl of media is removed and 100 μl of freshDifferentiation Media is added. On Wednesday 100 μl of media is removedand 100 μl of fresh Differentiation Media is added. On Thursday 100 μlof media is removed and 100 μl of fresh Differentiation Media is added.On Friday 100 μl of media is removed and 100 μl of fresh DifferentiationMedia is added. On the following Monday plates are prepared for thecalcium assay as follows: Plates are washed once with 10 mM Tris, HCl pH7-8. Working under a fume hood, 200 μl of 0.5 N HCl is added per well.Plates are then frozen at −80° C. Just prior to measuring calcium, theplates are freeze-thawed twice, and then trituration with a multichannelpipette is used to disperse the contents of the plate. The contents ofthe plate is then allowed to settle at 4° C. for 30 minutes at whichpoint an appropriate amount of supernatant is removed for measuringcalcium using a commercially available calcium kit. An exemplary andnot-limiting kit is Calcium (CPC) Liquicolor, Cat. No. 0150-250, StanbioLaboratory, Boerne, Tex.

In this cell based assay, sclerostin inhibits one or more of thesequence of events leading up to and including mineral deposition (i.e.sclerostin inhibits mineralization). Thus, in experiments wheresclerostin is included in the particular cell culture experiment, therecombinant sclerostin is added to the media starting on the firstThursday and every feeding day thereafter. In cases where ananti-sclerostin antibody is being tested for the ability to neutralizesclerostin, i.e., allow for mineralization by neutralizing sclerostin'sability to inhibit mineralization, the antibody is added to the mediastarting on the first Thursday and every feeding day thereafter. Theantibody is preincubated with the recombinant sclerostin inDifferentiation media for 45-60 minutes at 37° C. and then this media isused for feeding the cells.

Described above is a 12-day mineralization protocol for MC3T3-E1-BFcells. Mineralization of the original MC3T3-E1 cells is inhibited byrecombinant sclerostin and this inhibition is blocked using ananti-sclerostin neutralizing antibody, e.g., an anti-sclerostin antibodycomprising CDRs of SEQ ID NO: 245-247 and 78-80. The cell-basedneutralization assay is further described in U.S. Pat. No. 7,592,429 at,e.g., Example 8 (hereby incorporated by reference for its description ofcell-based neutralization assays).

Bone Specific Alkaline Phosphatase Assay—

An exemplary bone specific alkaline phosphatase assay is described inInternational Patent Publication No. WO 2008/115732 and U.S. Pat. No.7,744,874 (hereby incorporated by reference for its description ofcell-based neutralization assays). An exemplary protocol is as follows.C2C12 cells (ATCC, CRL 1772) are plated at 3000-5000 cells/well in a96-well tissue culture plate in MEM medium supplemented with 5% fetalcalf serum. The plate is incubated at 37° C. in 5% CO₂ overnight. Theantibody is diluted in 0.5× Wnt3a-conditioned medium (prepared asdescribed in WO 2008/115732) to various final concentrations. The mediumis removed from the plated cells and a pre-mixedantibody-BMP4-sclerostin solution (human or cynomologous monkey) isadded (150 μl), providing an antibody final concentration of 30 μg/ml to0.5 μg/ml, a final BMP-4 concentration of 25 ng/ml, a final sclerostinprotein concentration of 1.0 μg/ml, and the conditioned medium is at0.5× concentration. The plate is then incubated at 37° C. in 5% CO₂ for72 hours. The medium is removed from the cells, which are washed oncewith PBS, and frozen and thawed three times alternating between −80° C.and 37° C. Alkaline phosphatase activity is measured by adding alkalinephosphatase substrate (1-step PNPP, Pierce #37621) (150 μl/well). Theplate of cells is incubated for 60 minutes at room temperature, at whichtime optical density (OD) is measured at 405 nm to determine alkalinephosphatase activity. IC₅₀ calculations may be performed using, e.g.,SigmaPlot Regression Wizard with a Sigmoid 4-parameter fit equation.

BMP2-Induced MC3T3 Cell Mineralization Assay—

An exemplary BMP2-induced mineralization assay in MC3T3 cells isdescribed in International Patent Publication No. WO 2009/047356 (herebyincorporated by reference for its description of cell-basedneutralization assays). Briefly, MC3T31b cells are seeded in 96-wellplates (e.g., 6×10³ cells/well or 2×10³ cells/well) in 100 μl assayculture medium (maintenance culture medium without G418) and incubatedfor three days to reach confluence. The assay culture medium is changedand compounds to be tested are added with 10 mM b-glycerophosphate and50 μM ascorbic acid. Prior to addition to the cells, sclerostin and acandidate antibody are pre-incubated on a separate plate for two hoursat room temperature. To the assay 96 well-plates, 2.1 or 2.8 nM BMP-2(R&D Systems, Cat #355-BM-010) is applied before applying thesclerostin-antibody mixture. Cells are incubated for 14 days. At the endof the incubation, cells are washed twice with 200 μl PBS/well, 50 μl of0.5 M HCl is added to each well, and plates are frozen at −20° C. for aminimum of 24 hours. Plates are thawed at room temperature for 2 hoursfor testing. Ten 10 μl of each well is transferred to a new plate andexposed to Calcium Working Solution (1:5) (2041). Optical density ismeasured after a 5-30 minute incubation period at 595 nm on a microplatereader. Absorbance is translated into microgram of calcium according toa standard curve, allowing determination of the extent of BMP-2-inducedmineralization.

Cell-Based Wnt Signaling Assay—

An exemplary cell-based signaling assay employing super top flash (STF)reporter protein is described in International Patent Publication No. WO2009/047356. HEK293 cells are transfected with pcDNA3+ (480 ng);SuperTopFlash (STF) (20 ng); and phRL-CMV (0.5 ng) for control wells andpcDNA-wnt1 (20 ng); pcDNA3+ (460 ng); SuperTopFlash (STF) (20 ng); andphRL-CMV (0.5 ng) for Wnt1 treatment wells. The plasmids are mixed with1.6 μl of Lipofectamine 2000 diluted into 50 μl of OptiMEM® andincubated for 30 minutes at room temperature prior to application to thecells. Once applied, the cells are incubated at 37° C. in 5% CO₂ forfive hours.

Antibodies are premixed with SOST to generate a series of dilutions. Oneml of medium for each dilution is prepared, and 450 μl is added to eachwell after removing transfection mix. The cells are incubated with theantibody-SOST mixtures for 18-20 hours. At the end of the incubation,medium is removed, and 300 μl of 1× Passive Lysis Buffer (Promega, Cat #E194A) is added to lyse cells. Luciferase activity is then measuredusing Dual-Glo Luciferase System (Promega, Cat # E2940) with 30 μl oflysates in duplicates. Typically, 30 μl of Dual-Glo luciferase (fireflyluciferase; for STF) and 30p1 of Dual-Glo Stop and Glo (Renillaluciferase; for transfection efficiency control) substrates is used.Luminescent signals are measured with Mithras LB940 instrument (BertholdTechnologies). The ratio of firefly to Renilla luciferases iscalculated. The final results are expressed by setting the value of Wnt1without SOST as 1. Additional details of the assay are provided inInternational Patent Publication No. WO 2009/047356.

Example 2

This Example illustrates the ability of a sclerostin inhibitor, namelyan anti-sclerostin monoclonal antibody (Scl-Ab), to treat a bone gapdefect in a primate subject. This Example also illustrates thattreatment with a sclerostin inhibitor, namely an anti-sclerostinmonoclonal antibody (Scl-Ab), for a period of 28 weeks increasedcortical area and thickness without inducing adverse effects such asincreased cortical porosity in the bone of primate subjects.

Segmental defects (gap size 0.5 cm) were created in the middle of theleft ulna in 26 cynomolgus monkeys (male, age 4-4.5 years). Aftersurgery, the left arm was immobilized with fiberglass during the entireexperiment period. The animals were separated into two treatment groups:Group A (n=10) and Group B (n=16). The monkeys were subcutaneouslyinjected weekly with either vehicle (Group A) or Scl-Ab (Group B), at adose of 30 mg/kg, starting immediately post-surgery and continued for 28weeks post-surgery.

Animal body weight was measured at week 1, 2, 4, 6, 8, 10, 12, 14, 16,18 and 20. Radiographs of the left arm were taken immediately aftersurgery and at week 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 28.Tetracycline (a bone label), calcein (green fluorescent dye that stainscalcification tissues) and alizarin (red fluorescent dye that stainscalcification tissues) were administered at 6-7.5, 18-19.5 and 26-27.5weeks, respectively. Histomorphometric analysis of non-fracture femurdiaphysis was analyzed after the 28 weeks of treatment.

At week 28, only 1 out of 10 (10%) of the monkeys in Group A (control)had fully bridged the defect. In contrast, 6 out of 16 (38%) of themonkeys in Group B (Scl-Ab treatment group) had fully bridged thedefect. These results show that sclerostin inhibition by Scl-mAb canfill in a void or gap between bone segments to successfully treat a bonegap defect in humans.

Results indicated that treatment with Scl-Ab increased cortical area andcortical thickness at the femoral diaphysis at week 28 in the treatmentanimals. See FIGS. 2A and 2B. Monkeys having received the Scl-mAbtreatment also showed an increased periosteal and endocortical boneformation rate. See FIGS. 3A and 3B. Importantly, treatment with theScl-mAb for a period of 28 weeks did not substantially increase corticalporosity at the femoral diaphysis compared to the control monkeys. SeeFIG. 4.

The combined results described in this Example demonstrate that Scl-Abtreatment is not only useful for the treatment of skeletal defects suchas bone gap defects, but also that administration of Scl-Ab for a periodof longer than 6 months increases bone formation and bone mass withoutadverse effects such as increased cortical porosity. Increased corticalporosity is associated with an increased risk of fractures and decreasedbone strength.

All of the references cited herein, including patents, patentapplications, literature publications, and the like, are herebyincorporated in their entireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred compounds and methods may be used andthat it is intended that the invention may be practiced otherwise thanas specifically described herein. Accordingly, this invention includesall modifications encompassed within the spirit and scope of theinvention as defined by the following claims.

1. A method for treating a bone gap defect in a subject, the methodcomprising administering to a subject suffering from a gap of at least0.5 cm between two segments of bone an effective amount of ananti-sclerostin antibody wherein the anti-sclerostin antibody is anantibody that demonstrates a binding affinity for sclerostin of SEQ IDNO: 1 of less than or equal to 1×10⁻⁷ M; wherein the anti-sclerostinantibody is administered over a treatment period lasting at least 28weeks resulting in bridging the gap between the two segments of bone anddoes not result in a substantial increase in cortical porosity in thebridged bone of the subject, and the subject is not receiving a bonegraft, bone scaffold, prosthesis or a metal stabilizer.
 2. (canceled) 3.The method of claim 1, wherein the bone gap defect is selected from thegroup consisting of a comminuted fracture, a non-union fracture, asegmental skeletal defect, a surgically created bone defect, asurgically treated bone defect, and a bone defect created from traumaticinjury to the bone or disease.
 4. The method of claim 3, wherein thebone gap defect is produced by removal of infected sections of bone orthe removal of cancer from the bone.
 5. The method of claim 1, whereinthe subject is undergoing oral or maxillofacial surgery.
 6. The methodof claim 1, further comprising administering a second bone-enhancingtherapeutic selected from the group consisting of parathyroid hormone, abisphosphonate, a RANKL antibody and a DKK-1 antibody.
 7. The method ofany one of claims 1-6, wherein the anti-sclerostin antibody isadministered in an amount of 30 mg/kg per week.
 8. The method of any oneof claims 1-7, wherein the anti-sclerostin antibody is administered oncea week for the duration of the treatment period.
 9. (canceled)
 10. Themethod of any one of claims 1-9, wherein the anti-sclerostin antibody isadministered subcutaneously.
 11. The method of any one of claims 1-10,wherein the anti-sclerostin antibody is an immunoglobulin comprising aheavy chain and a light chain. 12-15. (canceled)
 16. The method of anyone of claims 1-11, wherein the anti-sclerostin antibody binds to asclerostin polypeptide comprising the amino acid sequence set forth inSEQ ID NO: 1, wherein said anti-sclerostin antibody binds to thesequence of at least one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, orSEQ ID NO:
 5. 17. The method of any one of claims 1-11, wherein theanti-sclerostin antibody binds to a sclerostin polypeptide comprisingthe amino acid sequence set forth in SEQ ID NO: 1, wherein saidanti-sclerostin antibody binds to the sequence of at least one of SEQ IDNO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO:
 73. 18. The methodof any one of claims 1-11, where the anti-sclerostin antibodycross-blocks the binding of at least one of antibodies Ab-A, Ab-B, Ab-C,Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10,Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20,Ab-21, Ab-22, Ab-23, and Ab-24 to sclerostin and/or is cross-blockedfrom binding to sclerostin by at least one of antibodies Ab-A, Ab-B,Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10,Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20,Ab-21, Ab-22, Ab-23, and Ab-24.
 19. The method of any one of claims1-11, wherein the anti-sclerostin antibody comprises a CDR-H1 of SEQ IDNO:245, a CDR-H2 of SEQ ID NO:246, a CDR-H3 of SEQ ID NO:247, a CDR-L1of SEQ ID NO:78, a CDR-L2 of SEQ ID NO:79 and a CDR-L3 of SEQ ID NO:80.20-21. (canceled)
 22. The method of any one of claims 1-11, wherein theanti-sclerostin antibody comprises CDRs of SEQ ID NOs: 20-25 ofInternational Patent Publication No. WO 2008/115732 (SEQ ID NOs:416-421), CDRs of SEQ ID NOs: 26-31 of International Patent PublicationNo. WO 2008/115732 (SEQ ID NOs: 422-427), or CDRs of SEQ ID NOs: 32-37of International Patent Publication No. WO 2008/115732 (SEQ ID NOs:428-433).
 23. The method of any one of claims 1-11, wherein theanti-sclerostin antibody comprises CDRs of SEQ ID NOs: 4, 15, 26, 37,48, and 59 of International Patent Publication No. WO 2009/047356 (SEQID NOs: 443, 454, 465, 476, 487, and 498, respectively).
 24. The methodof any one of claims 1-11, wherein the anti-sclerostin antibodycomprises the amino acid sequence of at least one of SEQ ID NOs:135-143, 153-161, or 171-179 of International Patent Publication No. WO2010/130830 (SEQ ID NOs: 745-753, 763-771, 781-789, respectively).